Derivatives of branch-chain lipophilic molecular and uses thereof

ABSTRACT

Novel phospho-derivatives of branched-chain lipophilic molecules useful for permeabilizing biological barriers and for inhibiting tumor growth are disclosed. Pharmaceutical compositions comprising said molecules and their uses are also disclosed.

FIELD OF THE INVENTION

The present invention relates to novel phospho-derivatives ofbranched-chain lipophilic molecules, to pharmaceutical compositionsthereof and to use thereof for increasing permeability of biologicalbarriers in a reversible and selective manner and for inhibiting tumorgrowth.

BACKGROUND OF THE INVENTION

A major limitation in the use of many drugs and therapeutic agents istheir inadequate ability to pass through biological barriers. Thispresents a serious problem especially for the treatment of diseases anddisorders in privileged sites such as the central nervous system (CNS).

The blood brain barrier (BBB), made up of specialized microvascularendothelial cells connected by tight junctions, is normally responsiblefor maintaining the homeostatic environment of the brain and protectingit from toxic agents and degradation products present in the circulatorysystem. However, in certain pathological situations, the presence of theBBB may interfere with the transport of therapeutic substances into thebrain, thus hampering treatment of central nervous system lesions,including tumors, infections, abscesses and degenerative diseases.

In a similar way, the presence of the blood tumor barrier (BTB)interferes with the delivery of chemotherapeutic agents into the tumor,thus decreasing drug bioavailability and preventing efficienttherapeutic effect where it is needed. The problem of insufficientaccess of the therapeutic agent to the diseased target is especiallysevere in the case of CNS tumors, and patients bearing malignant braintumors have poor prognoses.

In order to achieve clinically useful concentrations of certain drugs atrestricted sites, it is often required to administer these compounds athigh systemic dosages. The high systemic concentrations, in turn, areassociated with adverse side effects and high levels of toxicity.

One strategy for attacking the problem involves altering the biophysicalcharacteristics of hydrophilic drug molecules, for example, by linkingthese drugs to a lipophilic carrier. Since drug permeability across suchbiological membranes depends on its lipophilicity, increasing thelipophilic nature of the compound should, theoretically, improve itsbioavailability and increase therapeutic effects. Such covalent polarlipid conjugates with neurologically active compounds for targeting aredisclosed in U.S. Pat. No. 5,827,819 to Yatvin et al.

Another approach to circumvent the BBB impermeability is by employingagents that transiently open the BBB and facilitate the entry of aparticular drug or agent into the brain. Agents such as mannitol havebeen shown to exert this desirable effect and have been employed in thedelivery of chemotherapeutic agents to malignant brain tumors (Hiesingeret al. (1986), Annals of Neurology, 19:50-59). The use of this kind ofhyperosmolar BBB disruption in brain tumor therapy has, however, beencontroversial since, in addition to the drug crossing the BBB, othermolecules such as neurotoxins are also permitted entry. This may accountfor the high incidence of stroke, seizures, immunological reactions andocular toxicity associated with treatment using osmotic opening methods.

A variety of other treatments have also been disclosed that increasepermeability of the blood brain barrier including: the use of bradykininagonists (WO 91/16355 of Alkermes) and certain other peptides (WO92/18529 of Alkermes); use of bacterial cell wall fragments (WO 91/16064of the Rockefeller Univ.) or the use of antibody to Bordetella pertussisfilamentous haemagglutinin or brain endothelial x-molecule (WO 92/19269of the Rockefeller Univ.). Certain fatty acids such as oleic acid havealso been reported to reversibly open the BBB (Sztriha and Betz (1991),Brain Res. 336: 257-262).

The usefulness of methods for reversibly increasing the permeability ofthe blood brain barrier prior to administration of diagnostic reagents(U.S. Pat. No. 5,059,415 of the Oregon Health Sci. U.) or therapeuticreagents (WO 89/11299 of the Oregon Health Sci. U.) have been disclosed.

It was previously disclosed by the inventors of the present invention,in International patent application publication number WO 99/02120, thatbranched fatty acids and certain lipophilic derivatives thereof areuseful for reversibly permeabilizing biomembranes. However, compoundswhich comprise a phosphate moiety have not been disclosed. Furthermore,it has not been disclosed that by modifying the branched fatty acids byaddition of a phosphate moiety it is possible to modulate the opening ofbiological barriers in a specific and differential manner.

Clearly, the compositions developed so far for permeabilizing biologicalmembranes and barriers produce severe side-effects. Therefore, there isan unmet need for providing effective and safe means for deliveringadequate quantities of therapeutic and diagnostic agents into restrictedsites.

Numerous compositions have been proposed for use in treating variouscancers, included among them are compounds comprising a hydrocarbonchain and a phosphocholine moiety. U.S. Pat. Nos. 4,837,023 and5,049,552, both to Eibl, disclose compositions and methods useful intreating cancer. The active material in these cases is the knownsubstance Hexadecylphosphocholine (HePC). However, according to thedisclosure in those patents, of all compounds tested only HePC possesseda practically useful anti-tumor action, while homologues with shorteralkyl radicals possessed no or much too low anti-tumor action andhomologues with longer alkyl radicals were much too toxic. The prior artneither discloses nor suggests any of the compounds which are thesubject matter of the present invention.

SUMMARY OF THE INVENTION

The present invention provides, in one aspect, a compound of the generalformula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   R1 and R2 are the same or different, saturated or unsaturated    aliphatic chain comprising from 2 to 30 carbon atoms;-   R3 is A-[CH₂]_(m)—B—[CH₂]_(n)—C—[CH₂]_(p)-D , wherein m, n and p are    each independently zero or an integer from 1 to 12, and A, B, C and    D are each independently selected from a covalent bond, amino,    amido, oxygen, thio, carbonyl, carboxyl, oxycarbonyl, thiocarbonyl,    phosphate, amino phosphate mono- di- and tri-amino phosphate group    with the proviso that no two oxygen atoms are directly connected to    each other;

Z₁ and Z₂ are the same or different, each may be absent or independentlyselected from a) hydrogen, sodium, lithium, potassium, ammonium, mono-,di-, tri- and tetra-alkylammonium, or b) together with the phospho groupform a phospho ester of glycerol, choline, ethanolamine, inositol,serine, mono- or oligosaccharide.

In one preferred embodiment, the compound of the general formula I is anα-branched fatty molecule wherein R1 and R2 are hydrocarbon chainshaving, respectively, 3 and from 12 to 16 carbon atoms.

According to another preferred embodiment, R3 of the compound of thegeneral formula I comprises mono- or di-ethylene glycol moiety.

Currently preferred compounds according to the invention are:

-   -   4-Hexadecyl phosphate (3,12-PO₄),    -   4-Octadecyl phosphate (3,14-PO₄),    -   4-Eicosanyl phosphate (3,16-PO₄),    -   8-Pentadecyl phosphate (7,7-PO₄),    -   4-Hexadecanoyloxyethyl phosphate (3,12-MEG-PO₄),    -   2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate (3,12-DEG-PO₄),    -   4-Hexadecanyloxyethyl phosphate (3,12-(ether)-MEG-PO₄),    -   2-(4′-Hexadecanyloxy)ethoxyethyl phosphate        (3,12-(ether)-DEG-PO₄),    -   4-Hexadecyl phosphocholine (3,12-PC),    -   2-(4-Hexadecanoyloxy)ethyl phosphocholine (3,12-MEG-PC),    -   2-(4-Hexadecanoyloxy)ethoxyethyl phosphocholine (3,12-DEG-PC),    -   2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphate        (3,12-O—C₁₀-DEG-PO₄),    -   2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphocholine        (3,12-O—C₁₀-DEG-PC),    -   4-Octadecanoyloxyethyl phosphate (3,14-MEG-PO₄),    -   2-(4′-Octadecanoyloxy)ethoxyethyl phosphate (3,14-DEG-PO₄),    -   4-Octadecanyloxyethyl phosphate (3,14-(ether)-MEG-PO₄),    -   2-(4′-Octadecanyloxy)ethoxyethyl phosphate        (3,14-(ether)-DEG-PO₄),    -   4-Octadecyl phosphocholine (3,14-PC),    -   2-(4-Octadecanoyloxy)ethyl phosphocholine (3,14-MEG-PC),    -   2-(4-Octadecanoyloxy)ethoxyethyl phosphocholine (3,14-DEG-PC),    -   4-Eicosanoyloxyethyl phosphate (3,16-MEG-PO₄),    -   2-(4′-Eicosanoyloxy)ethoxyethyl phosphate (3,16-DEG-PO₄),    -   4-Eicosanyloxyethyl phosphate (3,16-(ether)-MEG-PO₄),    -   2-(4′-Eicosanyloxy)ethoxyethyl phosphate (3,16-(ether)-DEG-PO₄),    -   4-Eicosanyl phosphocholine (3,16-PC),    -   2-(4-Eicosanoyloxy)ethyl phosphocholine (3,16-MEG-PC),    -   2-(4-Eicosanoyloxy)ethoxyethyl phosphocholine (3,16-DEG-PC),    -   10-(4′-Hexadecanoyloxy)decanyl phosphate,    -   10-(8′-Pentadecanoyloxy)decanyl phosphate,    -   2-[2′-(2″-Propyleicosanoyloxy)-ethoxy]ethyl Phosphate        (3,18-DEG-PO₄), and    -   2-(2′-Propyleicosanoyloxy)ethoxy ethylphosphocholine        (3,18-DEG-PC).        Currently most preferred compounds are:    -   2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate, monosodium salt,    -   2-(4′-Hexadecanoyloxy)ethoxyethyl phosphate, diosodium salt,    -   2-(4′-Hexadecanyloxy)ethoxyethyl phosphate,    -   2-(4-Hexadecanoyloxy)ethyl phosphocholine, and    -   2-(4-Hexadecanoyloxy)ethoxyethyl phosphocholine.

Compounds of the invention are useful for increasing permeability ofbiological barriers. Thus, in another aspect, the invention providespharmaceutical compositions comprising an effective amount of a compoundof the general formula I depicted above, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention may further comprise apharmaceutically effective amount of a biologically active agent. In onepreferred embodiment, the biologically active agent is a therapeuticagent. The therapeutic agent may be selected from, but is not limitedto, anti-tumor, anti-viral, anti-microbial, anti-fungal,anti-inflammatory, neuroprotective agents and bioactive peptides andproteins.

In another preferred embodiment, the pharmaceutical compositions of theinvention further comprise a diagnostic agent.

The pharmaceutical compositions are useful for facilitatingadministration of biologically active molecules, for example therapeuticand diagnostic agents, into tissues and organs, in particular atprivileged sites which are protected by biological barriers.

In one particular embodiment, the pharmaceutical compositions are usefulfor increasing drug delivery across the blood retinal barrier (BRB),blood brain barrier (BBB) and blood tumor barrier (BTB). Thepharmaceutical compositions, in accordance with the invention, may alsobe useful for increasing permeability of other biological barriers,thus, for example, facilitating absorption through the skin, cornea,conjunctival, nasal, bronchial, buccal, vaginal and the gastrointestinalepithelium, and across the blood testis barrier and blood kidneyinterphase.

The pharmaceutical compositions may be administered by oral, parenteralor topical administration or by regional perfusion, enema or intra-organlavage. Preferably the pharmaceutical compositions of the invention areintra-arterially or intra-thecally administered.

In yet another aspect, the present invention provides methods forincreasing permeability of biological barriers. These methods compriseexposing said barriers to an effective amount of a compound of thegeneral formula I, or pharmaceutically acceptable salt thereof, thusenabling or increasing permeability of the biological barrier.

In still another aspect, the present invention provides a method foradministration of a biologically active agent into a privileged site ororgan comprising exposing said site or organ to said biologically activeagent in the presence of an effective amount of a compound in accordancewith the invention, thus enabling or increasing the penetration and/oraccumulation of the biologically active agent in the privileged site ororgan.

The privileged site or organ may be selected from, but is not limitedto, the spinal cord, brain, eye, testis, glands and tumors.

In still another aspect, the present invention provides a method fortreatment of a tumor comprising administering to a patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising as an active ingredient a compound of the generalformula I in accordance with the invention. Said tumor may be selectedfrom, but not limited to, carcinoma (e.g. breast, colon, rectal andbladder carcinomas), glioma (e.g. astrocytoma), neuroblastoma,retinoblastoma, intraocular malignancy, lymphoma, leukemia, sarcoma andmelanoma. The tumor may be a primary or secondary tumor.

The invention further provides a method for treatment of a centralnervous system disease or disorder comprising administering to a patientin need thereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound of the general formula I, or apharmaceutically acceptable salt thereof, in combination with atherapeutic agent. The therapeutic agent may be included in the samepharmaceutical composition comprising the compound of the generalformula I, or in a separate composition.

In a preferred embodiment the treated disease in the central nervoussystem is a brain tumor and the therapeutic agent is an anti-cancerdrug. In another preferred embodiment the treated disease is anophthalmologic disease or disorder, for example, cystoids macular edema(CME), Age-related macular degeneration (ARMD), intraocular infections,intraocular inflammations and intraocular malignancies.

In still a further aspect, the present invention provides a method forincreasing accumulation of a diagnostic agent in an organ protected by abiological barrier comprising administering to an individual saiddiagnostic agent in combination with an effective amount of a compoundof the general formula I as defined above, thus increasing accumulationof the diagnostic agent in the organ protected by a biological barrier.The diagnostic agent may be included in the same pharmaceuticalcomposition comprising the compound of the general formula I, or in aseparate composition.

In one preferred embodiment, said organ protected by a biologicalbarrier is the central nervous system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph correlating the various chain lengths ofα-branched fatty acids of the 3,n-type, with the potency of thesecompounds in extravasation of Evans blue-albumin complex into rat brain.

FIGS. 2A-C depict Evans blue accumulation in brain sections of ratsbearing bilateral gliomas following uni-lateral administration of acompound as follows: 10 μM 3,12-DEG-HPO₄Na (FIG. 2A), 40 μM 3,12-Na(FIG. 2B) or mannitol 25% (FIG. 2C).

FIGS. 3A-C depict fluorescein sodium salt (F—Na) angiography of theposterior part of a rat eye as recorded at 30 seconds and 10 minutesfollowing administration of F—Na either alone (FIG. 3A), or incombination with 3,12-DEG-HPO₄Na (FIG. 3B) or vehicle (FIG. 3C).

FIGS. 4A-B depict F—Na accumulation at the anterior part of the eye asrecorded 10 minutes after F—Na administration accompanied with either3,12-DEG-HPO₄Na (FIG. 4A) or vehicle (FIG. 4B) following the proceduredescribed in Example 26.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds, compositions and methods forincreasing permeability of biological barriers and for inhibiting tumorgrowth.

A desirable agent for facilitating delivery of therapeutic or diagnosticagents into restricted sites (e.g. the brain, eye, testis etc.) is onethat is capable of permeabilizing the limiting biological barrier.However, preferred permeabilization has to be accomplished in adifferential fashion without producing unacceptable degree of sideeffects. For example, in the case of treating a brain tumor, it would beadvantageous to use an agent that permeabilizes the BTB to a muchgreater extent than the BBB in the intact neighbouring brain tissue.Specific permeabilization, in this case, enables preferentialaccumulation of toxic drugs in the pathological tissue while causingminimal or no damage to the surrounding healthy brain tissue. Similarconsiderations apply for other biological barriers. For example, it isdesirable to have an agent that is capable of increasing delivery ofdrugs or other beneficial molecules across skin and intestine barriersin a transient and specific fashion.

The present invention concerns novel compounds and is based on theunexpected finding that these compounds may increase permeability ofbiological barriers in a reversible and selective manner. According tothe teaching of the present invention, the novel compounds are of thegeneral formula I as defined hereinabove or pharmaceutically acceptablesalts thereof.

In the specification, the compounds of the general formula I will becollectively referred to as “DP—BFAs” or interchangeably by the generalname phospho-derivatives of branched chain lipophilic molecules and inshort “P—BFA”. Branched chain lipophilic molecules not bearing aphosphate moiety will be referred to as “BFA” or “carbo-BFA”.

In one preferred embodiment the P—BFAs are phospho-derivatives ofα-branched lipophilic molecules of the general structure R1(R2)-CH—. Inother embodiments according to the present invention, the P—BFAs arephospho-derivatives of branched lipophilic molecules of the generalstructure R1(R2)-CH—[CH₂]_(m)— wherein m is from 1 to 12, thus referringto ω-branched lipophilic molecules branched, for example, at position β(m=1), γ (m=2) etc.

Specific DP—BFAs will be referred to, hereinafter, by their particularR1 and R2 that represent, respectively, the number of carbon atoms onthe side- and main-chain of the branched chain lipophilic molecule.

Compounds of the general formula I, wherein R3 is mono-ethyleneglycol ordi-ethyleneglycol will be referred to as R1,R2-MEG-PO₄ andR1,R2-DEG-PO₄, respectively. In some cases, the bond linking thebranched chain moiety and the adjacent chemical group is specificallyindicated as, for example, in 3,12-O—-C₁₀-DEG-PO₄, 3,12-(ether)-DEG-PO₄etc. Compounds of the general formula I wherein Z, is choline will bereferred to as R1,R2-PC.

In accordance with the teaching of the present invention, it ispossible, by varying the various components in the structure of thecompounds of the general formula I, to achieve fine-tuning in stabilityof the molecules and their permeabilization effect on biologicalbarriers. For example, varying the number of the carbon atoms in thealkyl groups R1 and R2 and the level of saturation affect the overallhydrophobicity of the molecule, thus enabling optimization of itsability to cross specific biological membranes.

Currently preferred compounds in accordance with the invention areDP—BFA molecules wherein the length of the carbon chain in R1 is from 2to 14, and the total number of carbon atoms in R2 and R3 together isfrom 6 to 26. Currently more preferred compounds are α-branched chaincompounds wherein R1 is 3 carbon atoms and R2 is from 12 to 16 carbonatoms. Preferred compounds of the general formula I comprise analiphatic chain having up to 20 carbon atoms in length counted from thephosphate group to the branching point of the branched lipophilicmolecule. This estimated number of carbon atoms in the R3 moiety of thegeneral formula I as defined above, is most suitable for effectivemembrane perturbation by the DP—BFA molecule and thus for eliciting ofthe desirable permeabilization effect. It should be noted that theabove-mentioned aliphatic chain may be a continuos hydrocarbon chain, ormay be interrupted by one or more hetroatoms selected from the group ofoxygen, sulphur, nitrogen and phosphorus atoms, as included in thedefinition of R3.

In one preferred embodiment in accordance with the invention, R3 of themolecule of the general formula I includes a carbonyl group linking thebranched chain moiety and at least one glycol moiety via an ester bond.In another preferred embodiment, R3 includes a covalent bond linking thebranched chain moiety and at least one glycol moiety via an ether bond.Under physiological conditions, ether bonds are generally lesssusceptible to enzymatic cleavage, therefore are expected to be morestable than ester bonds.

Another factor that may affect the ability of DP—BFA compounds topermeabilize various biological barriers is the polarity of thedifferent chemical groups in the molecule. This also may contribute tothe fine tuning of the differential permeabilization effect.

Preferred compounds of the invention are in salt forms, being eithermono- or di- salt compounds of the general formula I. Suitable salts mayinclude any pharmaceutically acceptable salt comprising a monovalent ordivalent counter ion which may be selected from, but is not limited to,Na⁺, Li⁺, K⁺, NH₄ ⁺, Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, mono-, di-, tri- andtetra-alkylammonium. More preferred compounds comprise a monovalentsalt. Particularly preferred are DP—BFA molecules in the sodium saltform, being either mono- or di-sodium salts.

The compounds of the invention may be prepared by chemical syntheticmethods well known in the art. Some of these methods are illustratedhereinafter in the Examples. Alternative procedures known to thoseskilled in the art may also be employed.

The compounds of the invention were found to be useful in disruptingtight cell junctions in vitro and in increasing permeabilization ofbiological barriers in vivo. Furthermore, the present invention is basedon the unexpected finding that certain derivatives of phospho-branchedchain lipophilic molecules have differential effects on opening of theBBB and BTB barriers. Accordingly, the various P—BFA compounds of theinvention may be useful in exerting permeabilization of biologicalbarriers while producing minimal toxicity and side effects.

Some DP—BFA compounds are selective in terms of being capable todifferentially permeabilizing biological barriers to molecules ofdifferent sizes. This characteristic may attribute to higher safetylevels of the phospho-BFA agents. Some DP—BFAs differ in the duration oftheir effect which may be proven as another beneficial factor in certaintherapeutic circumstances wherein transient selective permeabilizationof a limiting barrier is desirable.

The compounds of the invention and pharmaceutical compositionscomprising them may enable or facilitate delivery of biologically activeagents across biological barriers.

The term “biologically active agents” is intended to encompass allnaturally occurring and synthetic compounds capable of eliciting abiological response, having an effect on biological systems or servingas indicative tools. The biologically active agents may be therapeuticor diagnostic agents.

Therapeutic agents may include, but are not limited to, anti-neoplastic,anti-proliferative, anti-inflammatory, neurological, antibacterial,anti-mycotic and antiviral agents. These compounds in combination withthe compounds of the invention are particularly useful in the treatmentof pathological conditions, diseases and disorders in restricted sites.For example, in the treatment of central nervous system and lesionsincluding tumors, infections, abscesses and degenerative disorders.

Diagnostic agents may include, but are not limited to, imaging agents,contrast agents and dyes. Examples of diagnostic agents includeradioactively labelled substances (e.g. Technetium-99m and Fluoride-18based agents) and contrast agents such as gadolinium based compounds.The compounds of the invention may be useful, for example, in methodsfor diagnosing and characterizing brain lesions. In this case, acompound of the invention used for increasing BBB permeability, may beco-introduced (either in the same or separate composition) with achemical agent that is labelled in such a way that it can be monitored.

The chemical agent to be monitored may be, for example, a generalindicator to brain cells or structures, or one that binds or accumulatesspecifically and exclusively in certain brain areas or brain lesions.The brain, then, may be analyzed to determine the presence of labellingagent. Analysis may be performed using scanning and imaging means knownin the art (e.g. magnetic resonance imaging techniques (MRI), positronemission tomography (PET) and Computed Tomography (CT)).

In other embodiments of the invention, the DP—BFA compounds may beuseful for enhancement of drug absorption in the intestines or throughthe skin. The biological barriers in the intestines and skin preventpassive diffusion of a number of substances across the gastrointestinalor skin epithelium thus preventing effective absorption of certainusefil chemicals, nutrients and drugs. This obstacle may be overcome byapplying the desirable useful chemicals, nutrients and drugs incombination with a DP—BFA compound.

It will be readily apparent to those of ordinary skill in the art that awide range of biologically active compounds are appropriate for use incombination with the branched lipophilic molecules of the generalformula L therefore are included within the scope of the invention ascompounds useful in pharmaceutical compositions or methods of treatmentor diagnosis in accordance with the invention. In accordance with theinvention, the P—BFA compound is to be administered in combination withthe desirable biologically active agent. It should be clarified that thedesirable biologically active agent may be included in the samepharmaceutical composition of the compound of the general formula I, ormay be administered in a separate composition. Preferably both activeagents, i.e. the compound of the general formula I and the desirablebiologically active agent(s), are administered simultaneously or withina short period of time from one another. The biologically activeagent(s) may be administered prior or subsequent to the administrationof the P—BFA compound, provided that it will be within a time windowsuch that the effect of the P—BFA on the biological barrier issufficient to facilitate the passage of the relevant agents into therestricted site.

It is also possible that the desirable biologically active agent is notadministered around the same time of the P—BFA administration, but isalready present in the blood (being either naturally generated in thebody or produced as a slow release drug). Some examples includebioactive peptides and proteins such as neurotransmitters, growthfactors, hormones, antibodies etc. In this case the requirement is thatthe permeabilization effect of the target barrier occurs whilesufficient amount of the desirable biologically active agent is presentto exert its biological effect.

The improved activity of the compounds of the invention inpermeabilization of biological barriers results in increasedbioavailability of administered drugs, thus extending the therapeuticusefulness of these drugs to conditions that do not respond to lowerdoses of drugs. This is especially relevant in treatment of diseases anddisorders in restricted sites, e.g. brain and the eye.

In addition, the compounds of the invention are advantageous inasmuch asthey enable decrease in the useful dosage of drugs and consequentlyreduction in undesirable systemic side effects. Furthermore, since themolecules of the invention enable selective permeabilization ofbarriers, they may preferentially increase drug uptake at a specificsite (e.g. tumor) and not in the neighboring cells and tissues.

While examining the permeabilization effect of the P—BFA in vivo intumor-bearing rats, it was surprisingly found that some of the testedcompounds showed remarkable cytotoxic effects on the tumor, demonstratedin their ability to significantly inhibit the growth of the treatedtumor. Thus, it was established that certain compounds of the inventionmay be useful as anti-cancer agents. Moreover, some of the compounds ofthe invention, have demonstrated differential cytotoxic activity whentested for their effect on various normal and tumur cells in vitro.

In accordance with the principles of the present invention, the variousDP—BFA molecules may be specifically tailored to suit specific targetsites and specific indications. For example, molecules of the inventionwere found to act in a differential manner in opening the BBB and BTBbarriers. In this case the permeabilization of the BTB by certainDP—BFAs is to a greater extent comparing to the effect on the BBB.Another advantageous characteristics of the effect of some DP—BFAmolecules, is the preservation of certain levels of discrimination as tothe compounds permitted to cross. Thus the DP—BFAs affect barrierpermeabilization in a more selective manner comparing to carbo-BFAs orhyperosmotic agents, such as mannitol. As a result, the P—BFA compoundsare expected to cause less toxic side effect in comparison to otherpermeabilizers known in the art. Furthermore, as their permeabilizationeffect was found to be reversible, the compounds of the invention mayalso be useful for chronic administration of drugs.

As mentioned in the specification and claims, an “effective amount” ofP—BFA refers to that amount of a compound of the invention which exertsthe desirable beneficial effect in accordance with the invention.According to one aspect of the invention, it is the amount of P—BFA thatsignificantly increases the permeability of a relevant barrier to amolecule of interest. Namely, the amount of P—BFA which increases thepermeability of the relevant barrier to allow sufficient quantities of amolecule of interest to cross the biological barrier so to exert itstherapeutic or prophylactic effect or allow diagnostic procedures.According to another aspect of the invention, it is the amount of P—BFAthat is therapeutically effective as anti-cancer agent. Namely, thatamount of P—BFA which inhibits uncontrolled cell growth.

The effective amount will be determined on an individual basis and willbe based, at least in part, on consideration of the individual's size,the specific disease, the severity of the symptoms to be treated, etc.Thus, the effective amount can be readily ascertained by a person ofskill in the art employing such factors and using no more than routineexperimentation.

The dose range and the regimen employed will be dependent on the routeof administration, the age, sex, health and weight of the recipient andon the potency of the particular DP—BFA and the relevant useful drug oragent administered. The skilled artisan will be able to adjust theDP—BFA compositions and dosage in order to obtain the desired durationand the degree of action.

The pharmaceutical compositions may be in a liquid, aerosol or soliddosage form, and may be formulated into any suitable formulationincluding, but is not limited to, solutions, suspensions, micelles,emulsions, microemulsions, aerosols, ointments, gels, suppositories,capsules, tablets, and the like, as will be required for the appropriateroute of administration.

Any suitable route of administration is encompassed by the inventionincluding, but not being limited to, oral, intravenous, intramuscular,subcutaneous, inhalation, intranasal, topical, rectal or other knownroutes. In preferred embodiments for the permeabilization effect on theCNS, the pharmaceutical composition of the invention is intra-arteriallyor intra-thecally administered. For use as an anti-cancer medication,the relevant pharmaceutical composition of the invention is preferablyadministered orally or intravenously or applied topically or by regionalperfusion, enema or intra-organ lavage.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES I. CHEMICAL EXAMPLES

For the sake of clarity, procedures for synthesis of particular P—BFAmolecules and salts thereof are exemplified below. However it should beunderstood that similar procedures are also applicable for synthesis ofother P—BFA molecules of the invention including, but not limited to,saturated and unsaturated branched chain molecules and compounds whereinR1 and/or R2 are aliphatic chains comprising a cyclic alkyl group(s).Various pharmaceutically acceptable salts of the P—BFA molecules couldalso be obtained including, but not limited to, sodium, potassium,ammonium and alkyl-ammonium salts and salts with divalent counter-ions.

All synthesized compounds were characterized by NMR, mass spectroscopyand element analyses.

Example 1 Synthesis of Alkyl Phosphates

Phosphates of the general formula RO—P(O)(OH)₂ were prepared. Rrepresents a branched-chain alkyl moiety of the R₁(R₂)—CH structurewhere R₁ indicates the number of carbons in the side alkyl chain and R₂indicates the number of carbons in the main alkyl chain.

The synthesis of RO—P(O)(OH)₂ molecules is a three-stage procedure. Inthe first stage the corresponding alcohol (R—OH) was prepared fromaldehyde and alkyl bromide using the Grignard reaction (Vogel's,“Textbook of practical organic chemistry”, Wiley, New York, pg. 531,(1996).)

In the second stage diphenyl phosphate ester was prepared from thealcohol and diphenyl phospochloridate:ROH+ClP(O)(OC₆H₅)₂+C₅H₅N→RO—P(O)(OC₆H₅)₂+C₅H₅N.HCl

In the third stage alkanyl dihydrogen phosphate was obtained byhydrogenation of the diphenyl ester.RO—P(O)(OC₆H₅)₂+2H₂→RO—P(O)(OH)₂+2C₆H₆

4-Hexadecanyl Diphenyl Phosphate

Diphenyl phosphorochloridate (4.0 g, 0.015 mole) was added slowly whileshaking to a solution of hexadecane-4-ol (2.4 g, 0.01 mole) in drypyridine (5 ml) at room temperature. The flask was stoppered and setaside for 48 hr.; then the contents were poured into ice-cold 1Nhydrochloric acid (100 ml). The heavy oil, which separated was extractedwith ether. The ethereal layer was washed with 1N hydrochloric acid (3times), 5% sodium hydrogen carbonate (5 times), and water (5 times).After being dried (MgSO₄), the ether was removed, and the residue waspurified by column chromatography (Petrol Ether (bp 30-60° C.): Ether,10:1). After evaporation of a solvent 3.5 g of liquid was obtained.Yield 74%.

4-Hexadecanyl Phosphate. (3,12-PO₄)

A suspension of platinum oxide (Adams catalyst) (0.32 g) in glacialacetic acid (20 ml) was shaken under hydrogen atmosphere untilabsorption ceased. The Adams catalyst was then washed well with 2Nhydrochloric acid, water, and finally glacial acetic acid, bydecantation. Solution of 4-Hexadecanyl diphenyl phosphate (3.2 g) inglacial acetic acid (40 ml) was added to the catalyst, and the solutionwas shaken under hydrogen until absorption ceased. The catalyst wasfiltered off and washed with chloroform. The solvents were removed fromthe filtrate in vacuo. The residue was crystallized from petroleum ether(bp 30-60° C.) and dried at 65° C. 2.01 g of final product was obtained.Yield 92%.

4-Hexadecanyl Disodium Phosphate. (3,12-PO₄Na₂)

4-Hexadecanyl phosphate (1 g, 0.0031 mol) was dissolved in ethanol (100ml). NaOH (0.25 g, 0.0062 mol) was added and the mixture was stirred for1 hr. and then evaporated. Ethanol (2×100 ml) was added and evaporated.Ether (100 ml) was added and evaporated. The residue was crystallizedfrom acetone (30 ml) and dried at 65° C. (15 mm. Hg). 0.9 g of finalproduct was obtained. Yield 79%.

¹H-NMR (CD₃OD): δ 0.89 (m, 6H), 1.27 (s, 22H), 1.58 (m, 4H), 4.22 (m,1H). MS (FAB): m/z 367.06 (M+H)⁺.

4-Hexadecanyl Monosodium Phosphate. (3,12-HPO₄Na )

4-Hexadecanyl phosphate (3.13 g, 0.0097 mol) was dissolved in ethanol(150 ml). NaOH (0.37 g, 0.0092 mol) was added and the mixture wasstirred for 48 hrs and then evaporated. Ethanol (2×150 ml) was added andevaporated. Ether (2×100 ml) was added and evaporated. The obtainedsolid was triturated with acetone (100 ml) and dried under 1 mm. Hgatmosphere overnight. 2.98 g of final product was obtained. Yield 87%.

¹H-NMR (CD₃OD): δ 0.87 (m, 6H), 1.25 (s, 22H), 1.51 (m, 4H), 4.12 (m,1H). MS (FAB): m/z 345.11 (M+H)⁺.

4-Octadecanyl Disodium Phosphate. (3,14-PO₄Na₂)

¹H-NMR (CD₃OD): δ 0.89 (m, 6H), 1.27 (s, 26H), 1.58 (m, 4H), 4.17 (m,1H). MS (FAB): m/z 395.20 (M+H)⁺.

4-Octadecanyl Monosodium Phosphate. (3,14-HPO₄Na)

¹H-NMR (CD₃OD): δ 0.90 (m, 6H), 1.28 (s, 26H), 1.56 (m, 4H), 4.14 (m,1H), MS (FAB): m/z 373.29 (M+H)⁺.

8-Pentadecanyl Disodium Phosphate. (7,7-PO₄Na₂)

¹H-NMR (CD₃OD): δ 0.90 (m, 6H), 1.30 (s, 20H), 1.60 (m, 4H), 4.12 (m,1H). MS (FAB): m/z 352.93 (M+H)⁺.

8-Pentadecanyl Monosodium Phosphate. (7,7-HPO₄Na)

¹H-NMR (CD₃OD): δ 0.90 (m, 6H), 1.30 (s, 20H), 1.60 (m, 4H), 4.12 (m,1H). MS (FAB): m/z 353.05 (M+Na)⁺.

Example 2 Synthesis of 2-(2′-Propyltetradecanoyloxy)ethyl Phosphate

This compound was prepared by the same procedures as described above inExample 1 using the alcohol: C₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—CH₂—O—. Thisalcohol was obtained from chloride anhydride of 2-Propyl pentanoic acidand ethylene glycol (Vogel's, “Textbook of practical organic chemistry”,Wiley, New York, pg. 698, (1996)).

2-(2′-Propyltetradecanoyloxy)ethyl Disodium Phosphate (3,12-MEG-PO₄Na₂)

¹H-NMR (CDCl₃): δ 0.87 (m, 6H), 1.24 (s, 22H), 1.54 (m, 4H), 2.33 (m,1H). 3.87 (m, 2H), 4.26 (m, 2H). MS (FAB): m/z 439.19 (M+H)⁺.

2-(2′-Propyltetradecanoyloxy)ethyl Monosodium Phosphate(3,12-MEG-HPO₄Na)

¹H-NMR (CDCl₃): δ 0.87 (t, 6H), 1.24 (s, 20H), 1.42(m, 2H), 1.56 (m,2H), 2.34 (m, 1H). 4.02 (m, 2H), 4.28 (m, 2H). MS (FAB): m/z417.19(M+H)⁺.

Example 3 Synthesis of 2-[2′-(2″-Propyltetradecanoyloxy)-ethoxy]ethylPhosphate

This compound was prepared by the same procedures as described above inExample 1 using the alcohol:C₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—CH₂—O—CH₂—CH₂—O—. This alcohol was obtainedfrom chloride anhydride of 2-Propyl pentanoic acid and diethylene glycol(Vogel's, “Textbook of practical organic chemistry”, Wiley, New York,pg. 698, (1996)).

2-[2′-(2″-Propyltetradecanoyloxy)-ethoxy]ethyl Disodium Phosphate(3,12-DEG-PO₄Na₂)

¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 20H), 1.44 (m, 2H), 1.59 (m, 2H)2.39 (m, 1H). 3.72 (m, 4H), 4.09 (m, 2H), 4.24 (m, 2H). MS (FAB): m/z483.03 (M+H)⁺

2-[2′-(2″-Propyltetradecanoyloxy)-ethoxy]ethyl Monosodium Phosphate(3,12-DEG-HPO₄Na)

¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 20H), 1.44 (m, 2H), 1.59 (m, 2H)2.39 (m, 1H). 3.72 (m, 4H), 4.09 (m, 2H), 4.24 (m, 2H). MS (FAB): m/z461.31 (M+H)⁺.

Additional compounds were prepared in an analogous way. For Example, thecompound 2-[2′-(2″-Propyleicosanoyloxy)-ethoxy]ethyl Phosphate wasprepared by using the alcohol:C₃H₇—C(C₁₈H₃₇)H—C(O)—O—CH₂—CH₂—O—CH₂—CH₂—O—. This alcohol was obtainedfrom chloride anhydride of 2-Propyl eicosanoic acid and diethyleneglycol.

2-[2′-(2″-Propyleicosanoyloxy)-ethoxy]ethyl Monosodium Phosphate(3,18-DEG-HPO₄Na)

¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 34H), 1.44 (m, 2H), 1.59 (m, 2H)2.39 (m, 1H). 3.72 (m, 4H), 4.09 (m, 2H), 4.24 (m, 2H). MS (FAB): m/z544.31 (M+H)⁺.

Example 4 Synthesis of2-{2′-[10″-(Hexadecyl-4-oxv)decyl-1-oxylethoxy}ethylphosphate monosodiumsalt (3,12-O—C₁₀-DEG-HPO₄Na)

This compound was prepared by the same procedures as described above inExample 1 using the alcohol:C₃H₇—C(C₁₂H₂₅)H—O—(CH₂)₁₀—O—CH₂CH₂—O—CH₂—CH₂—OH—. This alcohol wasobtained in a two-step procedure: First, reacting 4-Hexadecanol tosylate(Vogel's, “Textbook of practical organic chemistry”, Wiley, New York pg.698, (1996)) with monosodium 1,10-decanediole to generate10-(Hexadecyl-4-oxy)decanol. At a second step, the alcohol2′-[10”-(Hexadecyl-4-oxy)decyl-ethyloxyethanol was prepared by reacting10-(Hexadecyl-4-oxy)decanol tosylate (generated as in step 1) withsodium 2-(2-hydroxyethyloxy)ethylate.

¹H-NMR (CD₃OD):δ 0.90 (m, 6H), 1.30 (s, 38H), 1.44 (m, 2H), 1.59 (m, 2H)3.15 (m, 1H). 3.37 (m, 4H), 3.45-3.62 (6H), 3,95(m, 2H). MS (FAB): m/z565.45 (M+H)⁺.

Example 5 Synthesis of 2(4-Hexadecanoxy)ethoxyethyl Phosphate

This compound was prepared by the same procedures as described above inExample 1 using the alcohol: C₃H₇—C(C₁₂H₂₅)H—O—CH₂—CH₂—O—CH₂—CH₂—O—.This alcohol was obtained from sodium 2-(2-hydroxyethyloxy)ethylate and4-Hexadecanol tosylate. (Vogel's, “Textbook of practical organicchemistry”, Wiley, New York, pg. 698, (1996)).

4-Hexadecanesulfonyl chloride

4-Hexadecanol (12.29 g 0.051 mol) and p-Toluenesulfonyl chloride (12 g0.063 mol) were dissolved Pyridine (100 ml) and stirred overnight atroom temperature. Dichloromethane (400 ml) was added. Dichloromethanesolution was then washed well with water, H₂SO₄ (3%), water, NaHCO₃ (3%)and water, dried with anhydrous magnesium sulphate (10 g). Afterevaporation of the solvent 20 g of crude 4-Hexadecanesulfonyl chloridewas obtained.

2(4-Hexadecanoxy)ethoxyethanesulfonyl chloride

Sodium (3.5g 0.15 mol) was added to Di(ethylene glycol) (140 ml 1.5 mol)at 60° C. in small pieces. To obtained solution 4-Hexadecanol tosylate(cmde 20 g) in THF (300 ml) was added at same temperature. Solution wasstirred at reflux for 6 hrs. Water (100 ml) was added to the mixture atroom temperature. The mixture was extracted with ethyl acetate (300 ml).After evaporation of the solvent the residue was purified by columnchromatography (Petrol Ether (bp 30-60° C.): Ether, 1:1). 4 g of2(4-Hexadecanoyl)ethoxyethyl was obtained. Yield 24% starting from4-Hexadecanol.

2(⁴-Hexadecanoxy)ethoxyethyl Disodium Phosphate (3,12-ether-DEG-PO₄Na₂)

¹H-NMR (CD₃OD): δ 0.9 (m, 6H), 1.28 (s, 22H), 1.43 (m, 4H), 3.30 (m,1H), 3.60 (m, 4H), 3.69 (m, 2H), 4.07 (m, 2H). MS (FAB): m/z455.45(M+H)⁺.

Example 6 Synthesis of2-(2′-Propyltetradecanoloxy)ethoxvethyl-phosphocholine

Phosphocholine of the formulaC₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—CH₂—O—CH₂—CH₂—O—PO⁻(O)—O—CH₂CH₂N⁺(CH₃)₃ wasprepared as follows:

2-(2′-Propyltetradecanoyloxy)ethoxyethylphosphocholine (3,12-DEG-PC)

To a cooled solution (0° C.) of2-(2′-propyltetradecanoyloxy)ethoxyethanol (15.8 g, 0.044 mol) andtriethylamine (10 ml, 0.075 mol) in dry ether (250 ml) was added2-chloro-2-oxo-1,3,2-dioxaphospholane (7 ml, 0.075 mol) in 200 ml of dryether. The mixture was stirred at room temperature for 2 hrs. Thecrystalline (C₂H₅)₃N.HCl that precipitated was filtered off, and thesolvent was removed in vacuum. The residue was dissolved in 500 mlsolution of trimethylamine (0.27M) in anhydrous acetonitrile andtransferred to a pressure bottle. The pressure bottle was kept for 48hrs in an oil bath at 60-65° C. The bottle was then cooled and opened.The solvent was removed, and the residue was purified by columnchromatography (CHCl₃:CH₃OH:H₂O, 1:9:1). The oil obtained afterevaporation of the solvent was lyophilized during 72 hrs at 65° C. 17.4g of slight yellow wax was obtained. Yield 75%.

¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29 (s, 22H), 1.46 (m, 2H), 1.59 (m,2H), 2.38 (m, 1H), 3.25 (S, 9H), 3.69 (m, 6H), 3.99 (m, 2H), 4.29 (m,4H). MS (FAB): m/z 524.6 (M+H)⁺.

The following compounds were synthesized by a process analogous to theabove-described procedure.

2-(2′-Propyleicosanoyloxy)ethoxy ethylphosphocholine (3,18-DEG-PC)

¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29 (s, 34H), 1.46 (m, 2H), 1.59 (m,2H), 2.38 (m, 1H), 3.25 (S, 9H), 3.69 (m, 6H), 3.99 (m, 2H), 4.29 (m,4H). MS (FAB): m/z 608.6 (M+H)⁺.

2-{2′-[10″-(Hexadecyl-4-oxy)decyl-1-oxy]ethoxy}ethylphosphocholine(3,12-O—C₁₀-DEG-PC)

¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29 (s, 34H), 1.46 (m, 2H), 1.59 (m,2H), 2.38 (m, 1H), 3.25 (S, 9H), 3.69 (m, 6H), 3.99 (m, 2H), 4.29 (m,4H). MS (FAB): m/z 608.6 (M+H)⁺.

Example 7 Synthesis of 2-(2′-Propyltetradecanoyloxy)ethylphosphocholine

Phosphocholine of the formulaC₃H₇—C(C₁₂H₂₅)H—C(O)—O—CH₂—CH₂—O—PO⁻(O)—O—CH₂CH₂N⁺(CH₃)₃ was prepared bythe same procedure as described above in Example 6, except that thecorresponding alcohol, i.e. 2-(2′-propyltetradecanoyloxy)ethanol, wasused instead of 2-(2′-propyltetradecanoyloxy)ethoxyethanol.

2-(2′-Propyltetradecanoyloxy)ethylphosphocholine (3,12-MEG-PC)

¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.29 (s, 22H), 1.46 (m, 2H), 1.59 (m,2H), 2.39 (m, 1H), 3.24 (S, 9H), 3.66 (m, 2H), 4.07 (m, 2H), 4.28 (m,4H). MS (FAB): m/z 480.7 (M+H)⁺.

Example 8 Synthesis of Alkylphosphocholines

Phosphocholine compounds of the formula RO—PO⁻(O)—O—CH₂CH₂N⁺(CH₃)₃ wereprepared by the same procedure as described above in Example 6, exceptthat R—OH was used as the alcohol in the initial step. R representsbranched chain alkyls of the R₁—C(R₂)H— type.

Hexadecanyl phosphocholine (3,12-PC)

¹H-NMR (CD₃OD): δ 0.91 (t, 6H), 1.28 (s, 22H), 1.57 (m, 4H), 3.21 (S,9H), 3.62 (m, 2H), 4.25 (m, 3H). MS (FAB): m/z 408.68 (M+H)⁺.

4Octadecanyl phosphocholine (3,14-PC)

¹H-NMR (CD₃OD): δ 0.9 (t, 6H), 1.28 (s, 26H), 1.57 (m, 4H), 3.21 (S,9H), 3.61 (m, 2H), 4.23 (m, 3H), MS (FAB); m/z 436.91 (M+H)⁺.

8-Pentadecanyl phosphocholine (7,7-PC)

¹H-NMR (CD₃OD): δ 0.92 (t, 6H), 1.32 (s, 20H), 1.59 (m, 4H), 3.23 (S,9H), 3.63 (m, 2H), 4.26 (m, 3H). MS (FAB): m/z 394.35 (M+H)⁺.

Example 9 Synthesis of ω-branched P—BFA

ω-branched P—BFA compounds are prepared by the same procedures describedabove in Examples 1 and 6 using the relevant ω-alcohols.

The preparation of ω-branched BFA is a six-stage synthesis. The startingreagents are 1-Bromo-ω-alcohol and n-Alkyl-m-alkylketone. The firststage is protection of the hydroxyl group of 1-Bromo-ω-alcohol byDihydropyran (DHP) following the procedure as described by Kociensid(“Protecting Group”; Georg. Thieme Verlag Stuttgart. N-Y. pg. 83-84(1994)).

THP denotes a Tetrahydropyranyl ether.

The second and third stages are standard Grignard synthesis of tertiaryalcohol (Vogel's, “Textbook of practical organic chemistry”, Wiley, NewYork, pgs. 475, 538, (1996)) as follows.

The fourth stage is reduction of tertiary hydroxy group of compound (IV)by ionic hydrogenation reaction following the procedure as described byCarey and Tremper. (JACS, v. 91, p. 2967, (1969))

The fifth stage is cleavage of the protection group off the obtainedco-branched alcohol (V) (“Protecting Group”; Georg. Thieme VerlagStuttgart. N-Y. pg. 83-84 (1994)).

The final stage, stage 6, is oxidation of the ω-branched alcohol to thecorresponded ω-branched BFA. This was carried out following theprocedure as described by Manger and Lee (Tetraehedron letters, v. 22,N. 18, p. 1655, (1981)).

Stage 6: RR′CH(CH₂)₁₀₇ OH+[O]→RR′CH(CH₂)_(ω)COOH

II. PHYSICO-CHEMICAL PROPERTIES Example 10 In Vitro LipophilicityMeasurements of DP—BFAs

The lipophilicity values of various derivatives of branched fatty acidswere estimated by comparing the solubility of these compounds in organicversus aqueous solutions. Octanol and physiological saline were used,respectively, as the organic and aqueous solutions. The partitioncoefficient (P_(c)) value, i.e. the octanol/saline distribution wasmeasured by the shake-flask technique. The results, i.e. mg/mlsolubility in octanol and water and the calculated LogP_(c), are shownin Table 1. TABLE 1 Octanol-saline partition coefficients (P_(c))Octanol Water Molecule* mg/ml mg/ml LogP_(c) 7,7 >50 >25 1.333,12 >20 >10 0.99 3,14 >20 >10 1.26 3,16 >20 9.4 1.26 7,7-PO₄ >10 >10 <53,12-PO₄ <5 <2.5 1.9 3,14-PO₄ >10 <0.5 1.11 3,12-MEG-PO₄ >10 >10 1.093,12-DEG-PO₄ >5 8 1.31 3,12-(ether)-DEG-PO₄ >5 >5 0.83 3,12-PC >10 >103,12-MEG-PC >10 >10 1.22 3,12-DEG-PC >50 >10 0.73*sodium salts of the branched fatty acids and their PO₄ derivatives

Example 11 Structure Function Correlations

In this study a correlation between the physico-chemical properties ofthe branched chain molecules and their biological effects was assessed.The study was aimed to find out whether there is a correlation betweenthe chain length of branched chain molecules and their potencies inEvans blue (EB) extravasation in rat brain (see Example 18 for theprocedure used in measuring EC₅₀ values). Various branched fatty acidsof the 3,n-type, wherein one branched chain is of 3 carbon atoms and thesecond hydrocarbon chain, n, is from 7 to 16 carbon atoms, were used.The results are graphically depicted in FIG. 1.

As can be seen in FIG. 1, BFAs 3,12, 3,14 and 3,16 were found to beactive in permeabilization of the BBB. Under the experimental conditionsemployed, the most potent compound among the tested molecules isBFA-3,12.

III. BIOLOGICAL EXAMPLES III-a) In Vitro Studies Example 12 ComparativeIn Vitro Toxicity Study

Various DP—BFA molecules were screened in cultured Chinese hamster ovary(CHO) AA8 cell line in order to establish their toxicity. LC₅₀ values,i.e. the concentrations causing death in 50% of cell population, werecalculated from dose response curves. LC₅₀ values of phospho-BFAs werecompared to those of the corresponding branched fatty acids(=carbo-BFAs).

Method

Lactate dehydrogenase (LDH) release was used as an assay for theintegrity of the cells membrane and, thus, for estimation of toxicity.Cells (2×10⁵/ml) were seeded (150 μl/well) in 96 well plates, inRPMI-1640 medium containing 10% FCS. After two days, tested DP—BFAcompounds or the control compound myristic acid were added to the platesin declining concentrations ranging from 500 to 1 μg/ml. Cells wereharvested after one hour. Forty-five minutes before harvesting, 16.5 μlof lysis solution was added to the first 6 wells in order to demonstratecomplete lysis and maximum lactate dehydrogenase release which indicatecell death. Other controls included, two blank wells and five wellscontaining cells with no additional drugs. Following centrifugation(1500 rpm, 5 min), 75 μl of the supernatant was transferred to a newplate with 45 μl of an LDH assay mix (Tox-7 kit, Sigma). After 10-20minutes at room temperature, absorbance was measured using an ElisaReader 490 nm.

Toxicity data obtained following 1 hour incubation with differentDP—BFAs is summarized in Table 2. Each experiment was performed intriplicate. TABLE 2 Toxicity of BFAs and their phospho-derivatives oncell cultures Tested compound LC₅₀ (μM) 7,7-Na 65 7,7-PO₄Na₂ 320 3,12-Na165 3,12-PO₄Na₂ 320 3,12-DEG-PO₄Na₂ 320

In general the cells were found to be more sensitive to carbo-BFA andless sensitive to the tested phospo-BFA derivatives. Sub-toxic doses forcarbo-BFA were estimated at between 30-100 μM following incubation for 1hour. Under the same conditions, sub-toxic doses for the testedphospho-BFA derivatives were estimated to range from 160 μM to more than640 μM.

Conclusion: In general, the phospho-BFAs were found to be around 2 to 5times less toxic than the corresponding carbo-BFAs molecules.

Example 13 Effects of DP—BFA on Epithelial Cell Junctions

Effects of the various BFA derivatives on cell-cell junctions wereinvestigated using the human adenocarcinoma cell line (A431). Thesecells have a polarized structure characteristic of intestinal cells.

In this study, quantitative changes in the subcellular distribution ofZO₂, a protein associated with tight junction complexes (Anderson et al.(1993) Current Opinion in Cell Biology 5: 772-778) was monitored.

A431 cells, cultured in DMEM with serum on glass cover-slips, wereincubated for one hour in the presence or absence of sub-toxic doses ofDP—BFA. The cells were fixed and permeabilized for 2 minutes with amixture of 3% paraformaldehyde and 0.5% Triton-X-100, and then furtherfixed for 20 min with 3% paraformaldehyde alone. The fixed cells wererinsed and incubated for 45 minutes at room temperature with rabbitpolyclonal antibody ZO₂ (Zymed Laboratories, Inc. USA), washed 3 timeswith PBS and incubated for 45 min with fluorescently labelled secondaryantibodies. Stained cover-slips were mounted in Elvanol (Mowiol 4-88,Hoechst, Frankfurt, Germany) before microscopic examination. Thedistribution of the immuno-fluorescently labelled ZO₂ protein wasmonitored.

A431 cells that were incubated for one hour with pervanadate, whichinhibits phosphotyrosine phosphatases and thus disrupts cell junctions,were used as a positive control.

The fluorescent imaging of ZO₂ indicated that DP—BFA has clear effectson disruption tight junctions of A431 cells.

Example 14 Effects of DP—BFA on Permeability of Epithelial CellMonolayer

The aim of this study was to assess the kinetics and overall effect ofthe DP—BFA compounds in modifying the permeability of an epithelial cellmonolayer, which served as an in vitro model system for biologicalbarriers and particularly for the intestinal barrier.

Two approaches were taken in order to determine the effect of DP—BFA onpermeabilization of biological barriers. One approach was to followtransport of a radioactive labelled compound across the epithelial cellmonolayer. A second approach was to monitor changes in electricalresistance as a measurement indicating the permeability level of thecell layer.

Epithelial cells are cultured on a membrane filter (0.4 micron, 1 cm²).The membrane with the epithelial cell monolayer is placed between twocompartments, a donor and a receiver chamber. The tested P—BFA compound,at predetermined non-toxic concentration, and ¹⁴C-sucrose radioactivetracer (1 Ci in 0.5 ml buffer) are added to the apical (donor) side.Samples of 0.5 ml each are collected from the basolaeral (receiver) sideat 10 minutes intervals for the 90 minutes duration of the experiment.Incubation is carried out in tissue culture medium containing 1% FCSwhile shaking at 70 rpm. The apical chamber and membrane are transferredinto a new receiver chamber at each sampling time point. This is done inorder to keep constant concentration of the tracer in the apical chamberand invariable liquid volumes in both chambers. The concentration ofradioactive tracer in the collected samples are estimated using a Triluxmicrobeta counter (Wallac, Finland). The rate of tracer transport iscalculated and expressed as Permeability Coefficient.

Integrity of the epithelial monolayer used in the experiment ismonitored at the beginning and end of incubation by measuring thetrans-epithelial electrical resistance (TEER) using millicell-ERC(Millipore).

A parallel set of experiments is conducted using the same experimentalsystem as described above but without the addition of the radioactivetracer. Trans-epithelial electrical resistance (TEER) was monitoredevery 10 minutes for a period of 90 minutes.

Conclusion: the fact that i) P—BFAs significantly increase the passageof sucrose across the epithelial cell monolayer, and ii) decrease theTEER of the monolayer, support the conclusion that P—BFA compoundspermeabilize biological barrier.

Example 15 Cytotoxic Activity of Various DP—BFAs on Normal and MalignantCell Types

The cytotoxic activity of various phospho-derivatives of branched-chainfatty acids (DP—BFAs) was assayed in vitro in cell cultures that includenormal and malignant cell types. The following cell systems were used:

-   Primary fibroblasts (human)-   Normal bone marrow (BM) cells (mouse)-   Primary bronchial epithelial cells (human, from Clonetics, cat. no.    CC-2541)-   Caco-2—Colon cancer cell line (human, ATTC, HTB-37)-   C6 glioma cell line (rat ATCC, CRL-2199)-   Neuro2a—neuroblastoma cell line (mouse, ATCC, CCL-131)-   SKBR-3—breast cancer cell line (human, ATTC, HTB-30)-   HL60—myeloid leukemia cell line (human, ATCC, CCL-240)-   U937—myeloid leukemia cell line (human)

Cells were seeded in a microtiter plate in MEM containing 2 mML-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin and 10% FCSat 37° C. The cultured cells were incubated, during their linear growthphase in the absence (control group) or presence of various DP—BFAs asindicated. The final concentrations of the tested DP—BFAs ranged from1.5 to 200 microMolars. At the end of the incubation period, which wasfive days for the bone marrow cells and three days for all the othercell lines, the cytotoxic effect of the added P—BFA on the cells wasestimated by using the calorimetric MTT assay. The MTT assay (Mosmann(1983) J. Immunol Methods 65: 55-63) measures mitochondrial reductaseactivity and serves for quantitative assessment of cellular viability.Drug concentration that causes 50% reduction in cell viability incomparison to the control group, is defined as EC₅₀. The EC₅₀ values ofthe tested DP—BFA compounds were calculated from dose response curvesestablished for each of the different assayed cell lines. The resultsare summarized in Table 3. Each EC₅₀ value is an average of EC₅₀ valuesderived from 1-5 independent experiments.

As can be seen from the results in Table 3, the various DP—BFAsmolecules were active to a different degree in their cytotoxic effect onthe different cells. Among the tested compounds, the most potentcytotoxic agents for malignant cells were 3,12-DEG-PO₄,3,12-(ether)-DEG-PO₄, 3,12-MEG-PC, 3,12-DEG-PC, 3,18-DEG-PC and 3,14-PC.Two of these compounds, 3,12-DEG-PO₄ and 3,12-(ether)-DEG-PO₄, werefound to have the lowest cytotoxic activity on normal epithelial cells.Another compound, 3,12-DEG-PC, was found to have the lowest cytotoxicactivity on normal fibroblasts and normal bone marrow cells. TABLE 3Toxicity of DP-BFAs on various cell lines Cell line C6 Normal primaryNormal bone Normal primary Caco-2 glioma Neuro2a SKBR HL60 U937fibroblasts marrow epithel Compound EC₅₀ (μM) 3,12-Na 100 76 130 683,12-PO₄—Na₂ >200 105 >200 90 3,12-MEG- >200 60 >200 75 PO₄Na₂ 3,12-DEG-40 38 58 60 25 65 62 >200 PO₄Na₂ 3,12-(ether)- 47 40 37 58 10 65 68 >200DEG-PO₄Na₂ 3,12-PC 150 62 47 3,12-MEG-PC 75 70 50 20 9 160 95 943,12-DEG-PC 113 95 134 40 11 6 170 166 90 3,18-DEG-PC 13 6 80 60 3,14-PC7 4 160 72Conclusions: Phospho-BFA compounds have demonstrated cell-type specificcytotoxic effects. Few of these compounds were shown as most potentcytotoxic agents when tested on malignant cell lines, while being muchless toxic when tested on normal cells from different tissues. Thesedata suggest that these compounds may be efficient anti-cancer agentswith low cytotoxic side effects.

Example 16 3,12-DEGPC Induces Activation of Caspase-3 and DNAFragmentation in a Neuroblastoma Cell Line

In order to further explore the possible mechanism underlying thecytotoxic effect exerted by the various DP—BFAs, two established markersfor apoptosis, caspase-3 activity and DNA fragmentation (Lincz (1998)Immunol. Cell Biol. 76: 1-19), were studied.

Neuro2a neuroblastoma cells (ATCC, CCL-131) were seeded in MEMcontaining 2 mM L-glutamine, 100 units/ml penicillin, 100 ug/mlstreptomycin and 10% FCS at 37° C. 24 hours later, when cells were inlogarithmic growth phase, 3,12-DEG-PC was added to a final concentrationof 25 μM, 50 μM or 100 μM. Cells grown in the presence of vehicle onlyserved as control group.

Assays for caspase-3 activity and DNA-fragmentation were performed oncell lysates obtained following, respectively, 6 and 24 hours incubationwith the drug. Caspase-3 activity was assayed using the fluorogenicsubstrate Ac-DEVD-AMC (Pharmingen, Becton Dickinson, cat. no. 66081U)and following the manufacture's instructions. DNA-fragmentation wasquantified by using the Cell Death Detection Elisa plus kit (Roche, cat.no. 1774-425) and following the manufacture's instructions.

The results are summarized in Table 4. The measured caspase activity isexpressed as percentage of the activity in the control plates normalizedto protein content. DNA-fragmentation is expressed as OD values at λ=405nm and is an average of readings from duplicate cell culture lysates.TABLE 4 Apoptosis markers in Neuro2a cells treated with 3,12-DEG-PCCaspase DNA activity fragmentation Added drug (% of control)(OD_(405 nm)) None (Control) 100% 0.030 3,12-DEG-PC, 25 μM 210% 0.0363,12-DEG-PC, 50 μM 540% 0.153 3,12-DEG-PC, 100 μM — 0.389

As can be seen from the results in Table 4, incubation of Neuro2a cellswith the compound 3,12-DEG-PC, caused about 5-fold increase in caspase-3activity, and a significant increase in DNA fragmentation. Similarresults were obtained when the compound was tested in another malignantcell line, caco-2, derived from colon carcinoma.

Conclusion: Apoptosis may be a possible mechanism for exerting thecytotoxic effect of phosphocholine derivatives of BFAs in certainmalignant cells.

III-b) In Vivo Studies Example 17 In Vivo Model System for Measuring theEffect of DP—BFA on BBB Permeabilization

Permeabilization of the BBB by DP—BFA was investigated in rat modelsystem by monitoring accumulation in the brain of two markers: a) Evansblue dye (EB), which rapidly binds in vivo with albumin (MW 70 kD) andis an indicator for paracellular transport; and b) Fluorescein sodiumsalt (F—Na), MW 376 D, which is transported via the cellular space, andenables monitoring transcellular transport. Upon BBB disruption theEvans blue-albumin complex irreversibly accumulates in the intercellularspace of the brain. The much smaller tracer fluorescein may betransported intracellularly to occupy both inter and intracellular spacein the brain.

Blood brain barrier permeabilization in Sprague-Dawley rats was inducedby brief exposure to DP—BFA. The animals were anesthetized withRampun-Imalgen and DP—BFA test compounds were administered via theexternal carotid artery retrograde to the brain (Smith, Q. R. Methods ofstudy. In: Physiology and Pharmacology of the Blood-Brain Barrier. EdBradbury M. W. B. Spinger-Verlag Berlin-Heidelberg-NY; 1992: 24-52). Thepterygo-palathina artery was ligated to avoid escape of infusedsolutions towards the external head vasculature. DP—BFA solutions wereinfused over a period of 30 seconds or half an hour, using a HarvardApparatus Syringe Pump. Blood flow through carotid communis artery wasinterrupted only at the time of infusion.

All tested DP—BFA molecules were prepared from stock solutions in eitherwater or ethanol by diluting 200-1000 fold into solution of isosmoticmannitol (5%) in Tromethamine buffer (Sigma) or PBS (pH 7.4)

The markers, Evans blue (EB, 25 mg/ml) and fluorescein (F—Na , 12.5mg/ml), in 1 ml solution, were injected intravenously immediatelyfollowing administration of the tested DP—BFA, or after a specific timeinterval if reversibility of action was investigated. Supportive i.v.infusion of F—Na (12.5 mg/ml-100 μL/min) was performed for 8 minutes.Brains were washed with saline solution (60 ml) 10 min after theintracarotid “flash”. Brain ipsilateral and contralateral hemispheresand tumors, where applicable, were homogenized separately with 50%trichloroacetic acid (TCA). The markers concentrations in the cortex andin tumors, where applicable, were determined spectrofluorometrically(Uyama, O. et al., (1988) J. Cereb. Blood Flow Metab. 8, 282-284:Abraham et al., (1996) Neurosci. Lett. 208, 85-88). The markers contentwas calculated as μg marker per one-gram brain tissue. Standard errorand Student's test for statistical significance were used.

Example 18 Effect of Branched Chain Fatty Acids of Various Chain Lengthson BBB Permeability

At the first stage of the study, BFA molecules of different chainlengths were screened for their effect in permeabilizing the blood brainbarrier (BBB).

The experiment was carried out on male Sprague-Dawley rats weighing250-320 g. The procedure described above in Example 17 was followed.

The effect of the tested compounds on the BBB opening was evaluated bytwo parameters:

a) efficacy—The degree of BBB opening estimated in terms of accumulationof Evans Blue albumin (μg EB/g brain tissue); and

b) potency—measured in terms of EC₅₀ and minimal effective concentration(MEC) values. EC₅₀ is the concentration of a tested compound producing50% of the maximal effect of Evans Blue extravasation. MEC is defined asthe concentration which enables accumulation of three times the amountof Evans blue accumulated in control animals, in our case a total of 3μg EB/g brain. The lower the EC₅₀ and MEC values are, the higher thepotency of the tested compound.

The results of evaluation of the effects of different DP—BFAs on BBBpermeability are presented in Table 5. TABLE 5 Effects of branched chainfatty acids on BBB permeability μg EB/g brain Molecule EC₅₀ (μM) MEC (atEC₅₀) 3,7-Na >450 — 0.8 3,10-Na ˜700 — 0.8 3,12-Na 20 ± 3 6.5 ± 0.7 32 ±4 3,14-Na 40 ± 5 9.1 ± 1.2 16.5 ± 2.5 3,16-Na 137 ± 17 18 ± 2  34 ± 57,7-Na 184 ± 21 132 ± 19   4.2 ± 0.6

As can be seen from the results in Table 5, there is substantialvariation in the degree of Evans Blue extravasation depending on theDP—BFA chain length. Those molecules with the shortest chain length arealmost completely ineffective in this model system. The molecules withthe highest efficacy were BFAs 3,12-Na and 3,16-Na, each elicitsaccumulation of more than 30 μg EB/g brain. In the experimental systemused, 3,12-Na is the most potent permeabilizing agent, having the lowestEC₅₀ and MEC values among the tested compounds.

Conclusion: Under the conditions of the experimental system employed,BFA 3/12-Na was found to be the most effective permeability enhancer interms of both potency and efficacy. Therefore, DP—BFA derivatives basedon BFA-3,12 were studied in more details.

Example 19 Effect of Different DP—BFAs on BBB Permeability

The effect of various DP—BFAs on BBB permeability was tested in themodel system of rat brains, as described above in Example 17. Efficacy,namely the level of the BBB opening was expressed as the amount of Evansblue accumulated in the brain (μg EB/g brain). Potency of the testedmolecules was estimated by their calculated EC₅₀ and minimal effectiveconcentration (MEC) values. TABLE 6 Effect of different DP-BFAs on BBBpermeability (in rat brains) μg EB/g brain Molecule EC₅₀ (μM) MEC (atEC₅₀) 3,12-PO₄—Na₂ 55 ± 1 3.3 ± 0.5 20 ± 3 3,14-HPO₄—Na  67 ± 11 6.2 ±0.9 10 ± 1 7,7-HPO₄—Na 150 — 1 3,12-MEG-PO₄Na₂ 18 ± 3 11 ± 2   5 ± 13,12-DEG-PO₄Na₂ 20 ± 2 3.3 ± 0.5 18 ± 3 3,12-(ether)-DEG-PO₄Na₂ 54 ± 57.3 ± 1.4   26 ± 5.2 3,12-PC  76 ± 14 23 ± 4  10 ± 1 3,12-MEG-PC 19.5 ±1.5 10 ± 1  15 ± 2 3,12-DEG-PC  70 ± 12 15 ± 2  14 ± 2 Mannitol 25% — 10± 2

As can be seen in Table 6, various DP—BFAs molecules promote the passageof the albumin bound to the Evans Blue tracer across the BBB. The testedcompounds differ in their potency and efficacy.

Under the conditions of this experiment, the most potent derivative of3,12-BFA was 3,12-DEG-PO₄Na₂ having an ED₅₀ value of 20 μM and minimaleffective concentration (MEC) of 3.3 μM. The most effective compounds inincreasing BBB permeability were 3,12-(ether)-DEG-PO₄Na₂, 3,12-PO₄Na₂and 3,12-DEG-PO₄Na₂. The accumulation levels of Evans Blue dye in ratbrain after treatment with EC₅₀ concentrations of the3,12-(ether)-DEG-PO₄Na₂, 3,12-PO₄-Na₂ and 3,12-DEG-PO₄Na₂ compoundswere, respectively, 26, 20 and 18 μg EB/g brain.

Example 20 Effect of DP—BFA on Transport of Evans Blue Bound Albumin andFluorescein Across the BBB

The effect of various DP—BFAs on BBB permeabilization to molecules ofdifferent sizes was examined in vivo in the model system of normal ratbrains.

The transport of two markers was followed: a) Evans blue bound toalbumin (MW 70 kD) as an indicator for paracellular transport; and b)Fluorescein sodium salt (MW 376 D) that represents small size molecules.

The Evans blue dye (EB) and Fluorescein sodium salt (F—Na) were infusedinto the artery carotis externa over 30 seconds according to theprotocol described above in Example 17. The test compounds were used attheir EC₅₀ concentrations. Control animals were treated with vehiclesolution.

Evans blue extravasation is expressed as the percentage of the totalamount of EB accumulated per one gram of brain tissue. The transport offluorescein into the brain, which is dependent on the serumconcentration of this molecule, is presented as percentage of the F—Nalevel in the serum.

The ratio of accumulation in the brain of F—Na to EB was calculated andthe results are presented in Table 7. TABLE 7 Effect of differentDP-BFAs on BBB permeability to EB-albumin and to Fluorescein % F—Na/ %EB/ Ratio * Molecule g brain g brain F—Na/EB control  0.3 ± 0.08 0.04 ±0.01 9.5 ± 1.6 Mannitol- 25% 2.4 ± 0.4  0.6 ± 0.21 4.0 ± 0.5 3,14-Na 3.8± 0.5  0.8 ± 0.07 4.7 ± 1.7 3,12-Na 6.4 ± 1.1 1.8 ± 0.2 4.9 ± 0.93,12-PO₄—Na₂ 8.2 ± 0.9 2.9 ± 0.6 5.9 ± 1.0 3,12-MEG-PO₄—Na₂ 5.0 ± 0.80.8 ± 0.1 8.4 ± 0.9 3,12-DEG-PO₄—Na₂ 9.0 ± 1.4 1.4 ± 0.2 9.0 ± 1.23,12-(ether)-DEG-PO₄Na₂ 4.4 ± 0.9   4 ± 0.8 1.1 ± 0.2 3,12-PC 3.0 ± 0.50.8 ± 0.1 7.8 ± 1.3 3,12-MEG-PC 5.2 ± 0.8 0.9 ± 0.2 6.9 ± 0.13,12-DEG-PC 2.9 ± 0.5 0.6 ± 0.1 6.8 ± 0.8* mean of individual ratios

As can be seen in Table 7, normal BBB (=control) is about 10 times morepermeable to Fluorescein than to EB-albumin complex. The hyperosmoticpermeabilizer, mannitol, increased BBB permeability to EB to a greaterextent than to F—Na (F—Nal EB ratio equals 4). Similarly the testedbranched fatty acids 3,14-Na and 3,12-Na showed F—Na/EB ratios of 4.7and 4.9, respectively.

The different phospho-derivatives of BFA affect BBB permeability to thetwo markers in a differential fashion. It was found that 3,12-PO₄increased permeability of the BBB to EB-albumin complex about 1.5 timesmore than the increase in permeability to Fluorescein. On the otherhand, it was found that 3,12-DEG-PO₄ increased permeability of the BBBin a more physiological manner, i.e. increased F—Na transport to thesame extent as EB extravasation. The F—Na/EB ratio in the rat brainsexposed to 3,12-DEG-PO₄ was around 9.

Conclusions: Various DP—BFA compounds affect BBB permeability indifferent manners. Some DP—BFAs, e.g. 3,12-(ether)-DEG-PO₄Na₂ and3,12-PO₄ (respective F—Na/ EB ratios of 1.1 and 5.9) induced selectiveopening of the BBB which favors transport of large molecules overmolecules with low molecular weight. In contrast, other molecules, e.g.3,12-DEG-PO₄, equally increased transport of both small molecules suchas F—Na and larger molecules such as EB-albumin.

Example 21 Duration of the Effect of DP—BFA on BBB Opening

The effects of DP—BFA on Evans Blue and Fluoroscein extravasation in ratbrain were examined at various time points following DP—BFAadministration.

Various DP—BFA compounds were infused, over a period of 30 seconds, intothe external carotid artery of Sprague Dawley rats, following theprocedure described above in Example 17. The tested compounds were usedat their EC₅₀ concentrations. Evans Blue and Fluoroscein extravasationin the brain was determined 10, 30, 60, 120 and 240 minutes followingDP—BFA administration. The content of Evans Blue (μg/g) and Fluoroscein(% of serum) in rat brain was calculated using an average of 2-3individual animals per time point.

The results obtained in the experimental system as described abovedemonstrate that the DP—BFA effect on the BBB permeabilization isreversible. Moreover, it is suggested that the BBB opening effects ofDP—BFA are relatively short lived. Most tested derivatives ofDP—BFA-3,12 maintain BBB permeability for less than one hour, havingD_(1/2) of around 30 minutes. D_(1/2) is defined as the duration of atleast 50% opening of the BBB using EC₅₀ concentrations of the testedcompounds. Two compounds, 3,12-PC and 3,12-MEG-PC, both comprising aphosphocholine moiety, have shown D_(1/2) which was 4 times longer thanthe other tested 3,12-BFA derivatives, i.e. D_(1/2) of around 120 min.

Conclusions: DP—BFA was shown to affect BBB opening in the rat brain ina reversible manner. Various DP—BFA have different durations of thepermeabilization effect.

Example 22 Toxicology and Safety Studies

DP—BFA molecules were evaluated for safety and toxicity. Increasingdoses of the tested compounds were administered intra-arterially (i.a.)into rats following the protocol described above in Example 17. Animalviability was monitored for 24 hours to evaluate lethal concentrations(LC). LC is defined as the minimal concentration that caused death.TABLE 8 Comparative safety data of different DP-BFAs (in rats) SafetyMolecule* LC(μM) MEC LC/MEC 3,12  40 ± 10  6.5 ± 0.7 6.1 3,14  78 ± 11 9.1 ± 1.2 8.6 3,16 431 ± 55 18 ± 2 23.9 7,7 360 ± 67 132 ± 19 2.73,12-PO₄  63 ± 12  3.3 ± 0.5 19.1 3,14-PO₄ 134 ± 18  6.2 ± 0.9 21.63,12-MEG-PO₄  80 ± 15 11 ± 2 7.5 3,12-DEG-PO₄ 30 ± 5  3.0 ± 0.5 103,12-PC  80 ± 16 23 ± 4 3.5 3,12-MEG-PC 15 ± 2 10 ± 1 1.5 3,12-DEG-PC300 ± 60 15 ± 2 20*sodium salt

Safety index is defined as the ratio between the lethal dose (LC) andthe minimal effective concentration (MEC), wherein MEC corresponds to acalculated concentration of the tested compound that elicitsaccumulation of three times the amount of Evans blue accumulated incontrol animals treated with vehicle only, i.e. total accumulation of 3μg EB. The higher the LC/MEC ratio is, the higher the safety index.

Conclusions: Introduction of a phosphate group increased safety index by2-3 folds without significantly hampering potency.

Example 23 In Vivo Model System for Measuring the Effect of DP—BFA onBTB Permeabilization (Tumor-Bearing Rats)

In order to study the ability of DP—BFA compounds to modulate bloodtumor barrier (BTB), a model system of C6 glioma-bearing rats, wasemployed.

Sprague Dawley (SD) rats were inoculated with C6 gliosarcoma cellsfollowing the procedure described by Bartis et al. (Exp Neurol (1996);142:14-28). A small burr-hole was made in the frontal scalp bone of therat fixed in a stereotaxis apparatus. Using a Hamilton syringe, 10 μl or5 μl of media, containing 2.5×10⁵ C6 gliosarcoma cells were inoculatedinto the frontal cortex. Coordinates were 1 mm posterior of bregma, 2.5mm—lateral, and 3 mm—depth. The needle remained in the place for 5 min.After removal of the needle, fascia and scalp were sutured. The ratswere checked 6-8 days after inoculation, when tumors of sufficient size(20-60 mg) had developed. The tumor was weighed after F—Na visualizationand dissection. Parts of the rats' brains were subject to histologicalexaminations and monitored for Evans Blue and Fluorescein uptake intoipsilateral tumour tissue, peritumoural tissue and contralateral tissue.

Results (not shown) obtained from experiments conducted on controltumor-bearing rats (not treated with the compounds of the invention)demonstrated that permeability of the blood tumor barrier (BTB) for EBand fluorescein is, respectively, about 2-fold and 4-fold higher thanthat found for the intact BBB.

Example 24 Effects of Various BFAs Molecules on BBB and BTB Opening(Study in Tumor-Bearing Rats)

The bilateral tumor model system of C6 glioma-bearing rats described inExample 23 above was employed for screening of various derivatives ofDP-BFA for their ability to permeabilize the blood brain barrier (BBB)in comparison to their effect on the blood tumor barrier (BTB). Theexperimental procedure described in Example 23 was followed except thatthe tumor-bearing rats were further treated with DP—BFA. The testedcompounds, at concentrations around their EC₅₀ values, were unilaterallyinfused into the external carotis artery over 30 seconds as described inExample 17. Animals treated with vehicle only serve as a control group.Permeabilization effect was quantitated by measuring accumulation ofalbumin bound Evans blue dye in tumor (T) versus non-tumor (NT) tissues.

Calculated efficacy values and specificity indices are summarized inTable 9. Specificity index is defined as the ratio between the levels ofEB accumulated in tumor and those accumulated in non-tumor brain tissues(T/NT). TABLE 9 Comparison of the BBB and BTB opening by various DP-BFAsBBB BTB EB μg/g Specificity EC₅₀ * EB μg/g Non-tumor Tumor/ Molecule(μM) Tumor brain Non-tumor 3,12-Na 22 ± 4 44 ± 8 43 ± 9 1.1 ± 0.33,12-PO₄ 10 ± 2 14 ± 6  2.5 ± 1.5 6.3 ± 1.3 3,12-MEG-PO₄ 22 ± 3 25 ± 321 ± 4 1.2 ± 0.2 3,12-DEG-PO₄  7 ± 2 34 ± 8  2.0 ± 0.2 22 ± 5  3,12-PC52 ± 7 17 ± 2 10 ± 2 1.8 ± 0.5 3,12-MEG-PC 20 12 6 2 3,12-DEG-PC 60 11 52 Mannitol  25% 12 ± 2  7 ± 2 2.1 ± 0.5* EC₅₀ values for tumor-bearing rats

As can be seen in Table 9, most tested compounds have shown similarlevels of EB accumulation in tumor and in non-tumor brain tissues, i.e.T/NT ratios of about 1 to 2. With mannitol (25%) the EB accumulation inthe tumor was about twice the accumulation in the non-tumor brain. Inthe experimental system used, the compound, 3,12-DEG-PO₄, showed highspecificity for opening the tumor blood barrier (BTB) manifested as EBaccumulation in tumor which was 22 times higher than the EB levels inthe non-tumor tissue (T/NT ratio=22).

Conclusion: 3,12-DEG-PO₄ showed the most specific effect on opening theBTB in C6 glioma tumors. This compound permeabilizes the blood tumorbarrier (BTB) of C6 gliomas to a greater extend than the normal bloodbrain barrier (BBB). DP—BFA derivatives may, thus, serve as agentscapable of specific and selective opening of the BTB.

Example 25 Unilateral DP—BFA Administration in SD Tumor-Bearing Rats

Evans Blue and Fluoroscein penetration into gliomas tumors was examinedfollowing unilateral infusion into the external carotis artery of either10 μM 3,12-DEG-HPO₄Na (FIG. 2A), 40 μM 3,12-Na BFA (FIG. 2B) or 25%mannitol (FIG. 2C). The relevant compound was infused into rat brains at9 to 12 days following inoculation of C6 glioma cells as described inExample 23.

It may be clearly seen from FIG. 2A that the Evans Blue stainingfollowing 3,12-DEG-PO₄ administration to rats with bilateral gliomas isonly in the tumor tissue in the ipsilateral hemisphere. On the otherhand, the carbo counterpart of the DP—BFA, i.e. BFA 3,12, and thehyperosmotic agent, mannitol, equally permeabilized tumor and non-tumorbrain tissues.

Example 26 Effect of DP—BFA on Blood Retinal Barrier (BRB)Permeabilization

Permeabilization of the blood retinal barrier (BRB) by DP—BFA wasinvestigated in rats by monitoring leakage of the fluorescent marker,fluorescein sodium salt (F—Na), from the blood vessels in the sclera.

Sprague-Dawley (SD) rats, weighing from 250 to 350 grams, wereanesthetized with a solution of Ketamin (100 mg/ml) and Xylasine (2%)injected at 0.1 ml/100 g body weight. The anesthetized animals wereinjected with 0.25 ml F—Na (12.5 mg/ml) into the jugular vein. Theretinal blood vessels were recorded with a 3-CCD color camera (teliCS5850) attached with a c-mount to the INAMI L-0960 microscope forophthalmic surgery. The microscope is equipped with a light source andan appropriate filter for F—Na detection. After 10 min the retina wascleared from the F—Na. At this point, 1.5 ml of 3,12-DEG-HPO₄Na (12.5μg/ml) was intra-arterially injected over a period of 30 seconds,accompanied with a second i.v. injection of F—Na. Pictures of the bloodvessels were taken at t=0 and 0.25, 0.5, 1, 2, 4, 8 and 10 minutesfollowing administration of the drug. The same experiment was repeatedin a second group of control rats, where 1.5 ml of vehicle (Tab-mannitol5%) was intra arterially injected instead of 3,12-DEG-HPO₄Na.

By monitoring the distribution of F—Na in the blood vessels at theposterior part of the animal eye, at the retina level, it wasdemonstrated that by 10 minutes following administration of F—Na alone,all the fluorescent dye was completely cleared from the area surroundingthe retinal blood vessels. However, i.v. injection of F—Na incombination with i.a. injection of 3,12-DEG-HPO₄Na, results in F—Naaccumulation around the blood vessels at the retinal level. This leakageof F—Na from the blood vessels, in the presence of 3,12-DEG-HPO₄Na, isan indication for the permeabilization of the blood retinal barrier. Inthe control animals, where only vehicle was administrated instead of theDP—BFA drug, the results were similar to those in the animals injectedwith F—Na alone, namely by 10 minutes following administration, nofluorescence could be detected in the blood vessels or at the areasurrounding them at the retinal level. The results depicted in FIGS.3A-C represent angiographia of the eye, namely pictures of the eye bloodsupply imaging taken at the retinal level as recorded at 30 seconds and10 minutes following administration of F—Na either alone (FIG. 3A), orin combination with 3,12-DEG-HPO₄Na (FIG. 3B) or vehicle (FIG. 3C).

In accordance with this observation, at 10 minutes following the F—Naadministration, higher amounts of F—Na could be detected in the vitreousof the animals treated with 3,12-DEG-BPO₄Na, than in the vitreous of thecontrol animals that were injected with the F—Na and vehicle only (FIGS.4A-B). The accumulation of the fluorescent signal at the vitreous is dueto the BRB permeabilization and F—Na leakage from the blood vessels atthe retinal level.

Conclusion: 3,12-DEG-BPO₄Na enables permeabilization of the blood retinabarrier, and results in F—Na accumulation in the retina and vitreous.

Example 27 Anti-Tumor Activity of DP—BFA

The anti-tumor effects of various P—BFAs were studied in the in vivomodel system described above in Example 17. Tested P—BFAs wereintra-arterially administered at the concentrations previouslydetermined as being effective in permeabilization of the BBB and BTB forEvans blue and Fluorescein extravasation. The experimental procedure fortumor inoculation, as described in Example 23, was followed.

On Day 3-4 following inoculation of the C6 gliosarcoma cells, rat werecannulated through external carotid artery and the tested P—BFAs wereinfused over 30 seconds, as described in Example 17, except that in thisexperiment no markers were administered. The treated rats weremaintained until Day 7-9, and then sacrificed. Coronal sections wereobtained, stained by eosin-hematoxylin, and reviewed histologically todetermine tumor volume. Animals treated with vehicle only serve ascontrol. Standard error and Student's test for statistical significancewere used. The results are summarized in Table 10.

As can be seen from the results in Table 10, P—BFA compounds exhibitcytostatic effect on glioma tumor growth in vivo. Under the experimentalconditions employed, the 3,12-DEG-PO₄ compound exhibited the mostpronounced anti-tumor effect. Other compounds, e.g. 3,12-MEG-PC, alsodemonstrated a significant anti-tumor activity, though to a lesserdegree. Conclusion: P—BFAs intra-arterially administered into C6glioma-bearing rats exhibit cytotoxic effect on the tumor. TABLE 10Anti-tumor effects of various P-BFAs on tumor growth P-BFA TumorConcentra- volume Molecule tions (μM) (mm³) ± SE P (to control) Control0  8.1 ± 2.22 — 3,12-MEG-HPO₄Na 30 4.2 ± 1.6 >0.1 3,12-PC 50 3.8 ±0.8 >0.1 3,12-MEG-PC 10 2.88 ± 0.6   <0.05* 3,12-DEG-PC 60 3.9 ±1.0 >0.1 3,12-DEG-HPO₄Na 5 1.5 ± 0.3  <0.01* 3,12-DEG-PO₄Na₂ 30 1.6 ±0.4   0.01*SE—standard error of the mean,*statistically significant difference.

Example 28 Anti-Tumor Effect of DP—BFA

The anti-tumor effect of DP—BFA was evaluated by monitoring tumor volumeand appearance in a model system of C6 glioma-bearing rats. SpragueDawley (SD) rats were inoculated with 2.5×10⁵ C6 glioma cells in 5 μlPBS as follows. Rats were mounted into a stereotactic head holder in aflat-skull position. After reflection of the periosteum, a burr hole waspreformed with a lrnm drill in the following coordinates: bregma −1.0,3.50 mm lateral from the midline on the left side, and at a depth of3.50 mm. The cell suspension was manually injected at a depth of 3.50 mmusing a 10 μl Hamilton syringe. The tumor bearing rats were treated 72 hpost inoculation with a single dose, ranging from 5 to 20 μM, of3,12-di-ethylenglycol phosphate (3,12-DEG-PO₄; sodium salt) or vehicle(iso-osmotic buffer, pH=7.4) infused into the external carotid arteryfor 30 sec. at a velocity of 3 ml/min. The pterigo-palatina artery waslegated, and the common carotid artery was clamped at the time ofinfusion.

Five days after the inoculation of the C6 tumor cells, the rats wereanesthetized with Ketamin/xylazin (1:1) 0.1 ml/100 grams body weight,perfiised with saline and fixed with 4% Formaldehyde solution. Brainswere removed and cryoprotected in a sucrose solution for 48 h. Thebrains were then sliced in coronal section, stained withHaematoxylin-Eosine and underwent pathology evaluation.

A significant decrease in the tumor size was observed in the treatedanimals in comparison to the non-treated animals. Tumor volume was8.1±2.2 mm³ (N=7) in the vehicle-treated animals and 1.6±0.2 mm³ (N=12)in the animals treated with 3,12-DEG-PO₄. In addition, themicroenvironment and the nature of the tumor growth were different.Vehicle treated tumor-bearing rats exhibited a remarkable local cellexpansion in the cortex, white matter and meninges. The C6 gliomasappeared irregular and highly infiltrative and showed a high tendency toinvade through the perivascular lymphatic spaces. In contrast, thetreatment with DP—BFA, given once on the 3rd day after cellsinoculation, significantly changed the character of the tumor growth.The C6 glioma invasiveness through the perivascular spaces wasdiminished and a solid tumor with defined border was observed. Somelimited invasion was found into the meninges around the inoculation areaand sub-ependima.

Conclusion: 3,12-DEG-PO₄ showed a significant anti-tumor effect asinhibiting tumor invasiveness and rate of growth. Thus, DP—BFA compoundsmay be useful in inhibiting spreading and invasiveness of tumors.

The skilled artisan will appreciate that the principles of the presentdisclosure are amenable to many embodiments and variations ormodifications, all of which are within the scope of the invention. Theexamples are intended to be construed as non-limitative, and the scopeof the invention is to be defined by the claims which follow.

1. A compound of the formula I:

or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 arethe same or different, saturated or unsaturated aliphatic chain havingfrom 2 to 30 carbon atoms; R3 is a covalent bond; and one of Z₁ and Z₂may be absent or is a hydrogen, and the other together with the phosphogroup forms a phospho ester of glycerol, choline, ethanolamine,inositol, or serine.
 2. The compound according to claim 1, wherein R1 is3 carbon atoms in length and R2 is from 12 to 18 carbon atoms in length.3. The compound according to claim 1, wherein R1 is propyl and R2 isdodecyl.
 4. (canceled)
 5. The compound according to claim 1, wherein thetotal number of carbon atoms in R2 is from 6 to
 26. 6. The compoundaccording to claim 1, wherein the total number of carbon atoms in R2 isfrom 12 to
 18. 7. The compound according to claim 1, wherein one of Z₁and Z₂ is absent, and the other together with the phospho group forms aphospho ester of choline.
 8. (canceled)
 9. The compound according toclaim 1 selected from the group consisting of: 4-Hexadecylphosphocholine (3,12-PC), 4-Octadecyl phosphocholine (3,14-PC), and4-Eicosanyl phosphocholine (3,16-PC). 10-14. (canceled)
 15. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the formula I, or a pharmaceuticallyacceptable salt thereof,

wherein: R1 and R2 are the same or different, saturated or unsaturatedaliphatic chain having from 2 to 30 carbon atoms; R3 is a covalent bond;and one of Z₁ and Z₂ may be absent or is a hydrogen and the othertogether with the phospho group forms a phospho ester of glycerol,choline, ethanolamine, inositol, or serine.
 16. The pharmaceuticalcomposition according to claim 15, wherein R1 is 3 carbon atoms inlength and R2 is from 12 to 18 carbon atoms in length. 17-42. (canceled)43. The pharmaceutical composition according to claim 15, wherein one ofZ₁ and Z₂ is absent, and the other together with the phospho group formsa phospho ester choline.
 44. The pharmaceutical composition according toclaim 15, wherein the compound of the formula I is selected from thegroup consisting of: 4-Hexadecyl phosphocholine (3,12-PC), 4-Octadecylphosphocholine (3,14-PC), and 4-Eicosanyl phosphocholine (3,16-PC. 45.The compound according to claim 1 which is 4-Hexadecyl phosphocholine(3,12-PC).
 46. The pharmaceutical composition according to claim 15,wherein the compound of the formula I is 4-Hexadecyl phosphocholine(3,12-PC).
 47. The compound according to claim 1 which is 4-Octadecylphosphocholine (3,14-PC).
 48. The pharmaceutical composition accordingto claim 15, wherein the compound of the formula I is 4-Octadecylphosphocholine (3,14-PC).
 49. The compound according to claim 1 which is4-Eicosanyl phosphocholine (3,16-PC).
 50. The pharmaceutical compositionaccording to claim 15, wherein the compound of the formula I is4-Eicosanyl phosphocholine (3,16-PC).
 51. A compound of the formula I:

or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 arethe same or different, saturated or unsaturated aliphatic chain havingfrom 2 to 30 carbon atoms; R3 is a covalent bond; and Z₁ is choline. 52.A method for treatment of a tumor comprising administering to a patientin need thereof an effective amount of a pharmaceutical composition ofclaim
 1. 53. The method according to claim 52, wherein the tumor is inthe central nervous system.
 54. The method according to claim 52,wherein the tumor is in the brain.
 55. The method according to claim 52,wherein the tumor is selected from the group consisting of carcinoma,glioma, neuroblastoma, retinoblastoma, lymphoma, leukemia, sarcoma andmelanoma.
 56. The method according to claim 52, wherein said tumor is aprimary or secondary tumor.